Bottom Line:
However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration.This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.

Introduction: Mesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.

Methods: Knocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect.

Results: We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.

Conclusions: We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.

Fig6: ASC migration is mediated by uPAR. a Scratch test assay was performed on ASC isolated from three different donors transfected with either si Neg or si uPAR and grown on type I dermal collagen in serum-free control medium or PDGF-AB-supplemented medium. Scale bars: 200 μm. b Quantification of ASC migration. Results expressed as percentages of wound closure at T = 22 h compared with T = 0 for each condition. Mean of three independent experiments ± SEM is represented (one donor per experiment), each performed in triplicate. *P <0.05. ASC adipose stem/stromal cells, si Neg nontargeting small interfering RNA, si uPAR uPAR-targeted small interfering RNA, PDGF-AB platelet-derived growth factor AB

Mentions:
We wondered whether uPAR was required for migration of MSC from other tissue origin such as ASC that can be isolated from adipose tissue. Migration assays were performed following PDGF-AB treatment and uPAR gene expression knockdown. ASC were transfected with control siRNA (si Neg) or uPAR targeted siRNA (si uPAR) and the knockdown efficiency was confirmed by quantitative RT-PCR and flow cytometry experiments that indicated, respectively, a 60 % decrease in uPAR mRNA expression (Figure S9A in Additional file 10) and a 90 % decrease in uPAR membrane protein expression (Figure S9B in Additional file 10). The scratch test assay was then conducted on type I dermal collagen in the presence or not of PDGF-AB. ASC migration showed a 30 % increase after 22 hours of treatment with PDGF-AB (Fig. 6a, b), while this increase observed under PDGF-AB treatment was inhibited when ASC were transfected with uPAR siRNA. On transwell dishes, PDGF-AB enhanced by sevenfold the number of migrated ASC and this effect was significantly reduced by uPAR siRNA (52 % reduction) (Figure S9C in Additional file 10). Similar results were thus obtained with ASC and BM-MSC, showing that PDGF-AB increased ASC migration on type I dermal collagen and that this effect was partially abrogated by knocking down uPAR gene expression. These results demonstrated that uPAR is required for PDGF-AB-induced migration of MSC from different tissue origin (bone marrow and adipose tissue).Fig. 6

Fig6: ASC migration is mediated by uPAR. a Scratch test assay was performed on ASC isolated from three different donors transfected with either si Neg or si uPAR and grown on type I dermal collagen in serum-free control medium or PDGF-AB-supplemented medium. Scale bars: 200 μm. b Quantification of ASC migration. Results expressed as percentages of wound closure at T = 22 h compared with T = 0 for each condition. Mean of three independent experiments ± SEM is represented (one donor per experiment), each performed in triplicate. *P <0.05. ASC adipose stem/stromal cells, si Neg nontargeting small interfering RNA, si uPAR uPAR-targeted small interfering RNA, PDGF-AB platelet-derived growth factor AB

Mentions:
We wondered whether uPAR was required for migration of MSC from other tissue origin such as ASC that can be isolated from adipose tissue. Migration assays were performed following PDGF-AB treatment and uPAR gene expression knockdown. ASC were transfected with control siRNA (si Neg) or uPAR targeted siRNA (si uPAR) and the knockdown efficiency was confirmed by quantitative RT-PCR and flow cytometry experiments that indicated, respectively, a 60 % decrease in uPAR mRNA expression (Figure S9A in Additional file 10) and a 90 % decrease in uPAR membrane protein expression (Figure S9B in Additional file 10). The scratch test assay was then conducted on type I dermal collagen in the presence or not of PDGF-AB. ASC migration showed a 30 % increase after 22 hours of treatment with PDGF-AB (Fig. 6a, b), while this increase observed under PDGF-AB treatment was inhibited when ASC were transfected with uPAR siRNA. On transwell dishes, PDGF-AB enhanced by sevenfold the number of migrated ASC and this effect was significantly reduced by uPAR siRNA (52 % reduction) (Figure S9C in Additional file 10). Similar results were thus obtained with ASC and BM-MSC, showing that PDGF-AB increased ASC migration on type I dermal collagen and that this effect was partially abrogated by knocking down uPAR gene expression. These results demonstrated that uPAR is required for PDGF-AB-induced migration of MSC from different tissue origin (bone marrow and adipose tissue).Fig. 6

Bottom Line:
However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration.This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.

Introduction: Mesenchymal stem cells (MSC) are well described for their role in tissue regeneration following injury. Migratory properties of endogenous or administrated MSC are critical for tissue repair processes. Platelet-derived growth factor (PDGF) is a chemotactic growth factor that elicits mesenchymal cell migration. However, it is yet to be elucidated if signaling pathways other than direct activation of PDGF receptor (PDGF-R) are involved in PDGF-induced cell migration.

Methods: Knocking down and co-immunoprecipitation approaches were used to evaluate urokinase-type plasminogen activator receptor (uPAR) requirement and its interactions with proteins involved in migration mechanisms, in human MSC induced to migrate under PDGF-AB effect.

Results: We demonstrated that uPAR activation and its association with β1-integrin are required for PDGF-AB-induced migration. This phenomenon takes place in MSC derived from bone marrow and from adipose tissue.

Conclusions: We showed that PDGF-AB downstream signaling requires other effector molecules in MSC such as the uPA/uPAR system and β1 integrin signaling pathway known for their role in migration. These findings provide new insights in molecular mechanisms of PDGF-AB-induced migration of human MSC that may be relevant to control MSC function and tissue remodeling after injury.